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Characterization of Human Metapneumovirus F Protein-Promoted Membrane Fusion: Critical Roles for Proteolytic Processing and Low pH
Rachel M. Schowalter, Stacy E. Smith, Rebecca Ellis Dutch
Rachel M. Schowalter
Department of Molecular and Cellular Biochemistry, University of Kentucky, Lexington, Kentucky 40536-0509
Stacy E. Smith
Rebecca Ellis Dutch
For correspondence: rdutc2@uky.edu
DOI: 10.1128/JVI.01287-06
Human metapneumovirus (HMPV) is a recently described human pathogen of the pneumovirus subfamily within the paramyxovirus family. HMPV infection is prevalent worldwide and is associated with severe respiratory disease, particularly in infants. The HMPV fusion protein (F) amino acid sequence contains features characteristic of other paramyxovirus F proteins, including a putative cleavage site and potential N-linked glycosylation sites. Propagation of HMPV in cell culture requires exogenous trypsin, which cleaves the F protein, and HMPV, like several other pneumoviruses, is infectious in the absence of its attachment protein (G). However, little is known about HMPV F-promoted fusion, since the HMPV glycoproteins have yet to be analyzed separately from the virus. Using syncytium and luciferase reporter gene fusion assays, we determined the basic requirements for HMPV F protein-promoted fusion in transiently transfected cells. Our data indicate that proteolytic cleavage of the F protein is a stringent requirement for fusion and that the HMPV G protein does not significantly enhance fusion. Unexpectedly, we also found that fusion can be detected only when transfected cells are treated with trypsin and exposed to low pH, indicating that this viral fusion protein may function in a manner unique among the paramyxoviruses. We also analyzed the F protein cleavage site and three potential N-linked glycosylation sites by mutagenesis. Mutations in the cleavage site designed to facilitate endogenous cleavage did so with low efficiency, and our data suggest that all three N-glycosylation sites are utilized and that each affects cleavage and fusion to various degrees.
Human metapneumovirus (HMPV) was identified in 2001 by careful analysis of samples from children with respiratory tract disease for which an etiological agent had not been identified (46). Since the discovery of HMPV, clinical studies have demonstrated that it is a causative agent of respiratory tract disease worldwide (17, 23, 27, 32, 38, 39), and examination of serological samples indicates that HMPV has been circulating in human populations since at least 1958 (46). Between 6 and 12% of children under age five with acute respiratory tract infections are positive for HMPV (18, 47, 48), the clinical symptoms of which closely resemble those seen with respiratory syncytial virus (RSV), ranging from coughing and wheezing to bronchiolitis and pneumonia (9, 10, 24). The cytopathic effect of HMPV has been reported to depend on the strain of HMPV, with some strains inducing syncytium formation similar to that of RSV, while other strains cause focal rounding and cell destruction (20). Sequence analysis places HMPV in the Pneumovirinae subfamily of the Paramyxoviridae family, as it is closely related to avian pneumovirus, a cause of severe respiratory infection in turkeys, as well as RSV, the most common cause of severe lower respiratory tract infection in infants and the elderly (16, 45).
As it is an enveloped virus, entry of HMPV into host cells requires the fusion of viral and cellular membranes. Paramyxovirus entry usually requires two viral glycoproteins, the fusion (F) and attachment (G, H, or HN) proteins (2, 14), and membrane fusion promoted by all paramyxovirus glycoproteins that have been examined takes place at neutral pH, with one possible exception (15, 21, 25). The mechanism of SER virus glycoprotein-induced fusion is controversial, as early studies indicated a requirement for low pH (43), but recent work has found efficient SER virus F protein-mediated membrane fusion at neutral pH (8). In addition to virus-cell membrane fusion, paramyxovirus glycoproteins also promote cell-cell fusion. Multinucleated giant cells, termed syncytia, can be found in tissues that have been infected by a variety of paramyxoviruses (26, 34). Cultured cells infected with RSV form syncytia, but examination of primary human airway epithelial cells infected with RSV suggests that syncytium formation by this virus may not be a common in vivo occurrence (50). Nevertheless, cell-to-cell fusion and the formation of syncytia are valuable tools for the study of fusion protein function. Expression of viral glycoproteins alone in tissue culture cells is sufficient to induce the formation of syncytia, thus permitting the careful examination of glycoprotein function in the absence of any other viral proteins.
The amino acid sequence of the HMPV F protein shares 33% sequence identity with the RSV F protein (45), and it appears to contain each of the features characteristic of type I viral fusion proteins (Fig. 1) (13). However, the HMPV F protein is only 10 to 18% homologous with the F proteins of paramyxoviruses outside the pneumovirus subfamily (45). The HMPV G protein, the putative attachment protein, shares no significant sequence similarity with other paramyxovirus attachment proteins, although it contains a high content of serine, threonine, and proline residues, which is also a feature of the RSV G protein (45). Whereas the attachment protein plays a key role in fusion of the Paramyxovirinae subfamily of the Paramyxoviridae family, its function in the Pneumovirinae subfamily now appears to be different. Recombinant HMPV, avian pneumovirus, and human and bovine RSV lacking the G protein can enter cells and proliferate, suggesting that the F protein alone can promote attachment and fusion (7, 31, 42, 44).
Schematic of the HMPV F protein. The F protein is proteolytically processed into two fragments, F1 and F2, which are disulfide linked. Arrows point to potential sites of N-linked glycosylation. The amino acid sequence upstream of the cleavage site is indicated along with the mutations made to this site. FP, fusion peptide; HRA and HRB, heptad repeats A and B; TM, transmembrane domain; WT, wild type.
Despite this notable distinction between the two branches of the Paramyxoviridae family, the F proteins of both appear to be structurally similar. Paramyxovirus F proteins are trimeric type I integral membrane proteins (13), and they are modified by N-linked carbohydrates, which are often required for protein folding or modulation of fusion (1, 11, 28, 29, 51). Furthermore, F proteins are synthesized as fusogenically inactive precursors (F0) that are subsequently cleaved to form a biologically active, disulfide-linked heterodimer, F1 + F2 (13, 22, 40). The great majority of paramyxovirus F proteins are cleaved by ubiquitous cellular proteases, such as furin or cathepsin L (35, 36). However, HMPV, like Sendai virus and human parainfluenza virus type 1, requires the addition of trypsin to efficiently propagate in cell culture, presumably to cleave the F protein (6, 46). Work performed by Schickli and coworkers suggests that trypsin does indeed process the HMPV F protein into a mature form (41). However, laboratory-expanded strains of HMPV in which the F protein underwent trypsin-independent cleavage were isolated, and the F protein residues that appeared to contribute to trypsin-independent cleavage were identified (41). Recently, the furin consensus sequence was introduced into the HMPV F protein cleavage site of a recombinant virus in order to examine the effects of trypsin-independent cleavage on tissue tropism and virulence (5). This F protein mutant was cleaved efficiently in the absence of trypsin, but infection was still restricted to the respiratory tract.
We chose to examine cell-cell fusion promoted by HMPV glycoproteins in transiently transfected cells in order to carefully dissect the basic requirements for membrane fusion promoted by this virus. In addition, we made mutations proximal to the putative F protein cleavage site and in potential N-linked glycosylation sites in order to understand the posttranslational modifications made to the HMPV F protein and the effects of these mutations on fusion. Using syncytium and luciferase reporter gene fusion assays, we show that the HMPV F protein can promote fusion in the absence of the G protein and that trypsin is indeed necessary to process the F protein into a fusogenic form. Surprisingly, we also found that fusion could be detected only when cells expressing the F protein were exposed to low pH, suggesting that this viral fusion protein may function in a manner unique among the paramyxoviruses.
Cell lines.Vero cells, BHK cells, and BSR cells (provided by Karl-Klaus Conzelmann, Max Pettenkofer Institut) were grown in Dulbecco's modified Eagle's medium (DMEM; Gibco Invitrogen, Carlsbad, Calif.) supplemented with 10% fetal bovine serum (FBS) and 1% penicillin and streptomycin. The media of BSR cells was supplemented with 0.5 mg/ml G-418 sulfate (Gibco Invitrogen) every third passage to select for the T7 polymerase-expressing cells.
Plasmids.HMPV F and G genes within the pGEM-3Zf(+) and pBluescript SK(−) vectors, respectively, were kindly provided by Ursula J. Buchholz (NIAID, Bethesda, Maryland). The HMPV F gene was released from pGEM-3Zf(+) and ligated into the pCAGGS mammalian expression vector following digestion with EcoR1 and Sph1. The HMPV G gene was released from pBluescript SK(−) and ligated into the pCAGGS mammalian expression vector following digestion with Sma1 and Xho1. HMPV G was moved from pBluescript SK(−) to the pGEM-4Z vector, using Pst1 and Sal1 restriction digestion sites. All HMPV F protein mutants were created while the gene was in pGEM-3Zf(+), using QuikChange site-directed mutagenesis (Stratagene). Then the F genes with mutations were subcloned into pCAGGS as described for the wild-type gene. All HMPV F, G, and mutant genes used in this study were sequenced in their entirety. The plasmids pCAGGS-SV5 F and pCAGGS-SV5 HN were provided by Robert Lamb (Howard Hughes Medical Institute, Northwestern University).
Antibodies.Antipeptide antibodies (Genemed Synthesis, San Francisco, Calif.) were generated using amino acids 12 to 26 of HMPV G and 524 to 538 of HMPV F.
Syncytium assay.Subconfluent monolayers of BHK or Vero cells in 6-well plates were transiently transfected with a total of 2 μg of DNA consisting of pCAGGS-HMPV F (1 μg) or an F protein mutant (1 μg) and pCAGGS-HMPV G (1 μg) or the empty pCAGGS vector (1 μg), using Lipofectamine Plus reagents (Invitrogen) according to the manufacturer's instructions. One exception is the "mock"-transfected BHK cells, which received 2 μg of the empty pCAGGS vector. The next morning, confluent cell monolayers were washed and incubated at 37°C in Opti-MEM (Gibco) with 0.3 μg/ml TPCK (l-1-tosylamide-2-phenylethyl chloromethyl ketone)-trypsin for 1 to 2 h (0.2 μg/ml TPCK-trypsin for BHK cells). Then the cells were rinsed once with phosphate-buffered saline (PBS; pH 7.2) before PBS of the indicated pH, buffered with 10 mM HEPES and 5 mM MES (2-(N-morpholino)ethanesulfonic acid hemisodium salt), was added. Cells were incubated for 4 min at 37°C under the pH conditions indicated in the figure, and then the media was replaced again with Opti-MEM with 0.3 μg/ml TPCK-trypsin. The pH pulse was repeated three more times (2 to 3 h apart) throughout the day. Vero cells were incubated for either a few hours at 37°C or overnight at 33°C in order to allow final cellular rearrangements to take place. Digital photographs of syncytia were then taken with a Nikon Coolpix995 camera mounted on a Nikon TS100 inverted phase-contrast microscope. Pictures of BHK cells were taken the same day, and BHK cells were maintained in Opti-MEM with TPCK-trypsin for only 1 h prior to each pH treatment. They were maintained in DMEM plus FBS the rest of the time.
Reporter gene fusion assay.Vero cells in 6-cm dishes were transfected with genes of viral glycoproteins or mutants, as indicated in the figure legends, and the T7 control plasmid (Promega) containing luciferase cDNA under control of the T7 promoter, using Lipofectamine Plus reagents (Invitrogen). The following day, Vero cells in one 6-cm dish were lifted from the plate surface with trypsin, resuspended in DMEM plus 10% FBS, and overlaid onto two 35-mm dishes of BSR cells. For pH titration experiments (see Fig. 3C), three 6-cm dishes of Vero cells were mixed and overlaid onto six 35-mm dishes, and each dish was then treated with a different pH solution. BSR cells constitutively express the T7 polymerase; thus, upon membrane fusion and content mixing of effector and target cells, luciferase is expressed. The combined cells were incubated at 37°C for 30 min. The cells were then rinsed once with PBS (pH 7.2) before PBS of the indicated pH buffered with 10 mM HEPES and 5 mM MES was added. The cells were incubated for 4 min at 37°C under the indicated pH conditions, the media was replaced again by Opti-MEM with 0.3 μg/ml TPCK-trypsin, and the cells were incubated at 37°C for 2 h. The cells were treated two additional times with buffered PBS 2 h apart while being maintained in Opti-MEM with trypsin between treatments. After the last pH treatment, the cells were incubated in DMEM with FBS at 37°C for 2 h. Finally, the cell lysates were analyzed for luciferase activity using a luciferase assay system (Promega) according to the manufacturer's protocol. Light emission was measured using an Lmax luminometer (Molecular Devices, Sunnyvale, Calif.).
Expression of proteins, metabolic labeling, and immunoprecipitation.Vero cells were transiently transfected with pCAGGS expression vectors using Lipofectamine 2000 (see Fig. 5A and B) or Lipofectamine Plus reagent (all others). At 18 to 24 h posttransfection, the cells were starved in methionine- and cysteine-deficient DMEM for 45 min and then metabolically labeled with Tran35S-label (50 to 100 μCi/ml; MP Biomedicals) for 1 h. The cells were washed once with PBS, and then incubated in serum-free DMEM ± 0.3 μg/ml TPCK-trypsin for 2 h. The cells were washed twice with PBS before being lysed with radioimmunoprecipitation assay buffer containing 100 mM Tris-HCl (pH 7.4), 150 mM NaCl, 0.1% sodium dodecyl sulfate (SDS), 1% Triton X-100, 1% deoxycholic acid, protease inhibitors (1 KalliKrein inhibitory unit of aprotinin, [Calbiochem, San Diego, Calif.] and 1 mM phenylmethylsulfonyl fluoride [Sigma, St. Louis, Mo.]), and 25 mM iodoacetamide (Sigma). The lysates were centrifuged at 136,500 × g for 10 min at 4°C, and the supernatants were collected. Antipeptide sera and protein A-conjugated Sepharose beads (Amersham, Piscataway, N.J.) were used to immunoprecipitate the F proteins as previously described (37). The immunoprecipitated F proteins were analyzed via SDS-15% polyacrylamide gel electrophoresis (SDS-PAGE) and visualized using the Typhoon imaging system.
Biotinylation.The cells were transfected in 6-cm dishes, starved, and metabolically labeled for 3 h as described above, except that trypsin was added to the labeling media. No chase was performed. The cells were washed three times with cold pH 8 PBS and incubated with 1 mg/ml EZ-Link Sulfo-NHS-Biotin (Pierce, Rockford, Ill.) diluted in pH 8 PBS for 10 min of rocking at 4°C and then 20 min at room temperature. The cells were washed three times again with pH 8 PBS and then lysed in radioimmunoprecipitation assay buffer as above. The proteins were immunoprecipitated as described above, and then beads were boiled twice in 10% SDS for 10 min to release the proteins. Fifteen percent of the total protein was removed for analysis, and the remaining 85% was incubated with immobilized streptavidin (Pierce) for 1 h at 4°C. Then biotin-labeled protein bound to streptavidin was pulled down and analyzed as described above.
PNGase F.Cells were transfected, starved, and metabolically labeled as described above and chased for 1 h in the absence of trypsin. PNGase F (N-glycosidase F; Sigma) digestion of the immunoprecipitated proteins was performed as previously described (37), except that 0.5 U of enzyme was used in the digestion. Samples were analyzed as described above.
In vitro coupled transcription/translation.A TNT Quick Coupled Transcription/Translation reaction (Promega) was assembled according to the manufacturer's instructions. The master mix, Tran35S-label, and pGEM-4Z-HMPV G or water (negative control) were combined. The mixture was incubated at 30°C for 60 min, then sample dye was added, and the product was analyzed via SDS-15% PAGE.
Requirements for HMPV F-promoted fusion.Fusion promoted by the HMPV F protein has yet to be examined in the absence of other viral proteins. To determine the basic requirements for HMPV F protein-promoted fusion, the HMPV F and G genes were subcloned into the pCAGGS vector, which supports a high level of transient expression in mammalian cells from a chicken actin promoter (33). Expression of the fusion and attachment proteins from most paramyxoviruses is sufficient to induce cell-cell fusion, resulting in the formation of syncytia, which are easily visualized microscopically. However, our initial experiments transfecting the HMPV F and G genes into multiple different cell types, including Vero, BHK, and LLC-MK2 cells, resulted in no formation of syncytia (data not shown). Since HMPV has been reported to require trypsin for efficient propagation (46), we repeated these assays in the presence of various concentrations of TPCK-trypsin. However, no reproducible syncytium formation was observed upon trypsin treatment of HMPV F- and G-expressing cells. Increased temperatures (45°C) and variations in the relative amounts of HMPV F and G transfected into cells also failed to stimulate syncytium formation, regardless of trypsin treatment (data not shown). Cell surface expression of the HMPV F and G proteins was confirmed by biotinylation of surface proteins following the metabolic labeling and trypsin treatment of cells expressing either HMPV F or HMPV G. Prior to the separation of the surface protein by streptavidin pull down, 15% of the total population of HMPV F or G, which was immunoprecipitated with antipeptide sera, was saved for comparison (Fig. 2A). Analyses of biotin-labeled surface populations of HMPV F confirm that trypsin treatment results in the presence of proteolytically processed F protein on the plasma membrane (Fig. 2A). However, a significant portion of the F protein population remained unprocessed by trypsin. Pulse-chase experiments with increasing concentrations of trypsin for various amounts of time did not result in increased processing, suggesting that a significant population of the F protein could not be cleaved (data not shown). Immunoprecipitated F protein also contained high-molecular-weight forms that are specific to cells transfected with HMPV F. These aggregates, which may correspond to higher oligomeric forms of the F protein, are quite stable. Increased concentrations of SDS or the addition of 6 M urea in the sample buffer and extended boiling of samples did not decrease the high-molecular-weight bands or increase the intensity of the bands corresponding to the predicted molecular weight of F0 (data not shown). The primary band in the total HMPV G population migrated at approximately 45 kDa, while the cell surface population also included a higher-molecular-weight form migrating at approximately 65 kDa. Both of these forms appeared unaffected by trypsin treatment (Fig. 2A). As the size of HMPV G as predicted by its amino acid sequence is considerably smaller than 45 kDa, we verified that the identified band was indeed HMPV G by use of in vitro coupled transcription/translation. HMPV G expressed from a pGEM-4Z plasmid with reticulocyte lysates migrated at a similar position to the product detected in cultured cells, suggesting that this ∼45 kDa species is indeed a product of HMPV G expression (Fig. 2B). The reason for the higher apparent molecular weight of HMPV G when visualized on an SDS-polyacrylamide gel is unclear. Biacchesi and colleagues noted a ladder of bands ranging from ∼13 to 90 kDa when HMPV G was immunoprecipitated from virions (7). This difference in our results may be due to various forms incorporated into virions or potential proteolysis.
Expression of HMPV F and HMPV G. (A) Biotinylation. Vero cells in 6-cm dishes were transfected with 4 μg pCAGGS-HMPV F or pCAGGS-HMPV G. The cells were metabolically labeled for 3 h with Tran35S-label ± 0.3 μg/ml TPCK trypsin prior to the biotinylation of surface proteins. Fifteen percent of the immunoprecipitated protein represents the "total" protein. The remaining 85% was subjected to pull down with streptavidin and represents the "surface" protein. The samples were resolved on a 15% SDS-polyacrylamide gel and visualized by autoradiography. Each side of the same gel was differentially contrast enhanced. (B) In vitro coupled transcription/translation of HMPV G ("in vitro"; right side) run side by side on a 15% SDS-polyacrylamide gel with HMPV G immunoprecipitated from transfected Vero cells ("in vivo"; left side).
The fusion proteins of some enveloped viruses, such as influenza virus HA, require low pH to induce membrane fusion between cells. Although low pH is normally not required for fusion promoted by paramyxovirus F proteins, we analyzed the HMPV F protein pH requirements by treating HMPV F- and G-expressing Vero cells (maintained the day after transfection in Opti-MEM with 0.3 μg/ml TPCK-trypsin, the maximum concentration of trypsin possible without visually impairing cell health) with a brief (4-min) pulse of pH 5-buffered PBS. Unexpectedly, syncytium formation could easily be visualized following this treatment, and additional pulses with low pH 18 to 36 h after transfection caused syncytium formation to increase (Fig. 3A). To determine if the HMPV G protein is essential for fusion, Vero cells were transfected with only HMPV F and empty vector. When treated with trypsin and low pH, the HMPV F-expressing cells formed syncytia at least as efficiently as the cells that were cotransfected with HMPV F and G (Fig. 3A). HMPV F did not promote syncytia in the absence of trypsin, even when treated with low pH (Fig. 3A). To verify that the result was not cell type specific, fusion was tested in BHK cells. Trypsin treatment combined with low pH also stimulated fusion by HMPV F in BHK cells, while neither trypsin nor low pH alone promoted syncytium formation above background in this cell type (Fig. 4). In contrast to Vero cells, HMPV G appeared to enhance HMPV F-promoted fusion in BHK cells, suggesting that the effect of G might be cell type specific (Fig. 4). Thus, our results demonstrate that efficient cell-cell fusion promoted by the HMPV F protein occurs only after proteolytic processing and exposure to low pH. Additionally, the HMPV G protein is not required for HMPV F-promoted membrane fusion, though it may enhance fusion promoted by the F protein in certain cell types.
Analysis of HMPV F-promoted cell-cell fusion. (A) Vero cells were transfected with pCAGGS-HMPV F (1 μg) and pCAGGS-HMPV G (1 μg) or the empty pCAGGS vector (1 μg). After 18 to 24 h, confluent cell monolayers were incubated in Opti-MEM ± 0.3 μg/ml TPCK-trypsin for 1 to 2 h and then washed and treated with buffered PBS of the indicated pH for 4 min. The cells were again incubated in Opti-MEM ± trypsin, and the pH pulse was repeated three more times throughout the day before the photographs were taken. (B) Vero cells were transfected with a total of 3.5 μg of DNA consisting of 1.5 μg luciferase cDNA, 1 μg pCAGGS-HMPV F or pCAGGS-SV5 F or empty vector, and 1 μg pCAGGS-HMPV G or pCAGGS-SV5 HN or empty vector. After 18 to 24 h, Vero cells were overlaid on BSR cells expressing the T7 polymerase. The cells were then treated and fusion was analyzed as described in Materials and Methods. The average of the results of four experiments, normalized to the luminosity detected from F-transfected, pH 5-treated cells, is shown. The error bars represent 95% confidence intervals. (C) Six-centimeter dishes of Vero cells were transfected with 1.5 μg luciferase cDNA and 1.5 μg pCAGGS-HMPV F. After 18 to 24 h, Vero cells were overlaid on BSR cells expressing the T7 polymerase. The cells were then treated and fusion was analyzed as described in Materials and Methods. The average of the results of four experiments, normalized to the luminosity of pH 5-treated cells, is shown. The error bars represent 95% confidence intervals. F&G, HMPV F and G proteins present.
Syncytia promoted by HMPV F in BHK cells. BHK cells were transfected with empty pCAGGS vector (2 μg) or pCAGGS-HMPV F (1 μg) and pCAGGS-HMPV G (1 μg) or empty vector (1 μg). After 18 to 24 h, confluent cell monolayers were incubated in Opti-MEM with 0.2 μg/ml TPCK-trypsin for 1 h, then washed and treated with PBS of the indicated pH for 4 min. The cells were then incubated in DMEM plus FBS for ∼1 h, then in Opti-MEM plus trypsin for ∼1 h. The pH pulse and change of media were repeated three more times before the photographs were taken. F&G, HMPV F and G proteins present.
Syncytium assays permit a determination of clear differences in fusion efficiency. However, accurate quantification of fusion in syncytium assays is difficult. Thus, reporter gene fusion assays were developed to allow quantitative measurements of HMPV F-promoted fusion. Vero cells transfected with viral glycoproteins and luciferase cDNA under control of the T7 promoter were lifted from the plate surface with trypsin (at concentrations that effectively cleave the HMPV F protein), and the cells were gently pelleted, resuspended in DMEM plus 10% FBS, and overlaid onto BSR cells, which constitutively express the T7 polymerase. After 30 min at 37°C to allow cell attachment, the mixed cell population was treated with pH 7- or pH 5-buffered PBS for 4 min. The cells were retreated with trypsin and exposed to pH 7 or pH 5 PBS two more times (2 h apart), the total cell population was lysed, and luciferase activity was analyzed.
In the reporter gene fusion assay with pH 7 pulses, no difference in fusion could be detected between cells transfected with only HMPV G and those transfected with HMPV F either alone or in combination with HMPV G (Fig. 3B). In contrast, there was a three- to fourfold increase in luciferase activity over background (HMPV G alone, pH 5) when cells transfected with HMPV F and G were exposed to pH 5 PBS. Interestingly, HMPV G appeared to have a negative impact on fusion, as cells transfected with only HMPV F induced a sevenfold increase in fusion over background (Fig. 3B). Biotinylation experiments showed that cell surface expression of HMPV F was not modulated by the coexpression of HMPV G (data not shown). To ensure that the increase in luciferase expression was due to the specific effects of low pH on the trigger of membrane fusion by HMPV F, fusion induced by simian virus 5 (SV5) glycoproteins was measured under the same conditions used to examine HMPV fusion. SV5 glycoprotein-induced fusion has been examined extensively, and fusion induced by these viral glycoproteins is not affected by low pH (8). As expected, our data showed that fusion induced by SV5 glycoproteins was not increased by treatment with pH 5 PBS (Fig. 3B), verifying that the effects of pH seen with HMPV were not due to nonspecific effects. Fusion induced by SV5 glycoproteins was always greater than fusion induced by HMPV F, which is to be expected if HMPV F can cause fusion only when treated with low pH, while SV5 F may cause fusion continuously.
Since fusion induced by HMPV F was stimulated by low pH, we next performed a reporter gene fusion assay in which cells were exposed to a range of pH conditions to determine the threshold or optimal pH for fusion induced by HMPV F. Fusion induced by HMPV F increased gradually as the pH was decreased, with maximum levels of fusion attained at pH 4.5 (Fig. 3C). Lower-pH treatments were not examined due to a lack of physiological relevance. Syncytium assays with HMPV F and G in Vero cells confirmed the results of the reporter gene assay. A gradual increase in fusion was apparent as the pH of the treatment decreased. However, a sharp difference between pH 5 and pH 4.5 in the syncytium assay was less evident (data not shown).
Mutation analysis of the HMPV F cleavage site.As we had established functional fusion assays for the HMPV F protein, we next created a series of mutations proximal to the putative cleavage site in the HMPV F protein to determine the role of the residues in proteolytic processing and fusion. In addition, we created an HMPV F protein with a recognition sequence for the endogenous protease furin to examine the effects of intracellular processing on HMPV F trafficking and membrane fusion promotion. The wild-type and cleavage site mutant amino acid sequences are displayed in Fig. 1. The cell surface expression of each of these mutants in the presence or absence of trypsin was examined by Tran35S-label metabolic labeling and biotinylation combined with immunoprecipitation and SDS-PAGE (Fig. 5A and B). Also, the ability of each mutant to induce cell-cell fusion was examined in syncytium and reporter gene assays (Fig. 6A to C).
Analysis of the surface expression of HMPV F mutants. Vero cells in 6-cm dishes were transfected with 8 μg pCAGGS-HMPV F or a mutant F protein. The cells were metabolically labeled for 3 h with Tran35S-label in the absence (A) or presence (B) of 0.5 μg/ml TPCK trypsin. Biotinylation and analysis were performed as for Fig. 2A, but only the surface populations are shown. Fwt, wild-type F protein.
Fusion promoted by HMPV F cleavage-site mutants. Syncytium assays in the presence (A) or absence (B) of trypsin are shown. Vero cells were transfected with 1 μg pCAGGS-HMPV F or a mutant F protein and 1 μg pCAGGS-HMPV G. The cells were treated (each with pH 5 PBS) and analyzed as for Fig. 3A. (C) Reporter gene assay of fusion in the presence of trypsin. Vero cells in 6-cm dishes were transfected with 1.5 μg luciferase cDNA and 1.5 μg pCAGGS-HMPV F or a mutant. After 18 to 24 h, the Vero cells were overlaid on BSR cells expressing the T7 polymerase. The cells were then treated and fusion was analyzed as described in Materials and Methods. The average of the results of three experiments is shown, and the error bars represent 95% confidence intervals. The graph is divided into two sections. The mutants on either side were analyzed on different days but always alongside the wild-type (WT) protein, and values were normalized to that of the wild type in each experiment.
The arginine at position 102, after which cleavage was proposed to take place, was mutated to an alanine (P1). Trypsin, which cleaves the C-terminals of basic residues, would not be expected to properly process this mutant. Indeed, this mutant was not cleaved in the presence or absence of trypsin (Fig. 5A and B), and no fusion could be detected (Fig. 6A and C). Therefore, the predicted F protein cleavage site is likely the correct site, and like other paramyxovirus F proteins, the HMPV F protein must be cleaved for membrane fusion to occur.
Next, the P2 residue (second amino acid upstream of the cleavage site) was changed from a serine to a proline, as it has recently been reported that certain laboratory-grown strains of HMPV acquired this mutation when propagated in the absence of trypsin (41). However, the F protein used in this study (CAN97-83) is most similar to a strain of HMPV (A2) used in the previous study that did not acquire the P2 mutation and could not grow in the absence of trypsin. Examination of the P2 mutant by SDS-PAGE and in fusion assays showed that it was not cleaved by endogenous proteases (Fig. 5A and 6B), suggesting that other amino acid changes are needed to promote trypsin-independent cleavage of this F protein. Subsequently, the glutamic acid found 10 residues upstream of the cleavage site was mutated to a lysine, both alone (E93K) and in combination with the P2 mutation (P2/E93K), as this mutation was also reported to enhance cleavage (41). However, these mutants could not promote fusion in the absence of trypsin (data not shown), and they were cleaved only with the addition of trypsin (Fig. 5A and B). Schickli et al. also found that a number of viruses containing the P2 mutation in F acquired an additional glutamine-to-lysine mutation in the P3 position when grown in the absence of trypsin (41). We created this Q-to-K mutation in the P3 position of F, both alone (P3) and in combination with P2 (P2/P3). The P2/P3 mutant promoted the formation of small syncytia in the absence of trypsin when exposed to low pH (Fig. 6B), and a low level of F protein cleavage was detected by surface biotinylation and SDS-PAGE (Fig. 5A). When only the P3 residue was mutated, fusion and cleavage in the absence of trypsin were not detected (Fig. 5A and 6B), suggesting that both the serine-to-proline change at P2 and the glutamine-to-lysine change at P3 were necessary for proteolytic processing by endogenous proteases. Still, cell-cell fusion induced by the P2/P3 mutant in the absence of trypsin was considerably less than that of the trypsin-treated wild-type protein (Fig. 6A and B). Furthermore, cell-cell fusion promoted by P2/P3 increased in the presence of trypsin (Fig. 6A and B), suggesting that inefficient cleavage rather than changes in F protein stability resulted in the decreased fusion. Finally, a mutant was created in which the second and third residues upstream of the cleavage site were changed to arginines, making all four residues upstream of the cleavage site arginine. This mutant (furin) is predicted to be cleaved efficiently by the trans-Golgi-resident enzyme furin, which has strong preference for R-X-K/R-R sequences. Cell surface expression of the cleaved F1 protein appeared to be slightly greater for the furin mutant than the P2/P3 mutant (Fig. 5A), although cleavage of the furin mutant was still quite low compared to other paramyxovirus F proteins that are normally cleaved by furin. Fusion induced by this mutant in the absence of trypsin was similar to that of the P2/P3 mutant (Fig. 6B), and syncytium formation by the furin mutant was also increased by trypsin treatment (Fig. 6A and B), suggesting that cleavage by endogenous proteases was less than optimal.
Fusion by each of the mutants, except P1, was stimulated by treatment with both trypsin and low pH, and fusion by each under these conditions was similar to or slightly less than that of the wild type (Fig. 6A and C). Notably, fusion induced by the furin mutant in the presence of trypsin was less than that of the wild type, indicating that, while these amino acid changes permitted endogenous cleavage they had a negative effect on fusion promotion. However, this may be a consequence of the reduced levels of cleaved protein on the cell surfaces (Fig. 5B). Only the mutants P2/P3 and furin promoted syncytium formation in the absence of trypsin (Fig. 6B and data not shown), but syncytium formation was very low, and fusion by these mutants in the absence of trypsin was not detected over background levels of fusion in a reporter gene assay (data not shown).
Analysis of N-linked glycosylation in HMPV F.The HMPV F protein contains three sites (N-X-S/T) with the potential to be N-link glycosylated in the endoplasmic reticulum. PNGase F treatment of immunoprecipitated HMPV F suggested that one or more of these sites was utilized, as a large shift in F0 was apparent on a reducing polyacrylamide gel following incubation with PNGase F (Fig. 7A). On the other hand, PNGase F treatment of HMPV G indicated that this protein was not N-link glycosylated. However, there is a higher-molecular-weight form of HMPV G detected only when surface populations are examined (Fig. 2A), which may be the product of carbohydrate modification. However, the decreased G protein left after overnight PNGase treatment was below the limit of detection in our assays. The asparagines of HMPV F with the potential to be glycosylated are found at positions 57, 172, and 353 (Fig. 1), and each of these asparagines was mutated individually to alanines. These mutants (N57, N172, and N353) were analyzed by SDS-PAGE to reveal changes in mobility, as this would indicate that the site mutated is normally glycosylated. Figure 7B demonstrates that all three potential sites are utilized for glycosylation, as F0 of each mutant appears roughly 2 kDa smaller than the wild type F0. Each mutant is expressed on the cell surface (Fig. 5B), indicating that none of the carbohydrates individually is required for folding in the endoplasmic reticulum and subsequent transport to the cell surface.
Analysis of HMPV F N-linked glycosylation. (A) PNGase F treatment of HMPV F and G. Vero cells in 35-mm dishes were transfected with 2 μg pCAGGS-HMPV F, pCAGGS-HMPV G, or empty vector. Cells were metabolically labeled with Tran35S-label for 1 h and chased for 1 h. Immunoprecipitated protein was incubated overnight with or without PNGase F, and samples were resolved on a 15% SDS-polyacrylamide gel and visualized by autoradiography. (B) Vero cells were transfected with 2 μg pCAGGS-HMPV F or a mutant. Cells were metabolically labeled with Tran35S-label for 1 h and chased for 2 h in serum-free DMEM ± 0.3 μg/ml TPCK-trypsin. Immunoprecipitated protein was analyzed as above. (C) Syncytium assay. Vero cells transfected with 1 μg pCAGGS-HMPV F or a mutant and 1 μg pCAGGS-HMPV G were treated (each with pH 5 PBS) and analyzed as for Fig. 3A. (D) Reporter gene assay. Vero cells in 6-cm dishes were transfected with 1.5 μg luciferase cDNA and 1.5 μg pCAGGS-HMPV F or a mutant. After 18 to 24 h, Vero cells were overlaid on BSR cells expressing the T7 polymerase. The cells were then treated and fusion was analyzed as described in Materials and Methods. The average of the results of seven experiments is shown, and the error bars represent 95% confidence intervals.
Removal of the carbohydrate at N57 found within F2 appeared to slightly reduce trypsin-mediated proteolytic processing compared to that of the wild type (Fig. 5B and 7B). The size of F1 does not change, as predicted for a loss of glycosylation in F2, and we were unable to detect F2 by autoradiography. This is likely a consequence of low levels of cleavage combined with the presence of only two cysteine and no methionine residues in F2. The residues N172 and N353 are located between the two heptad repeat regions of F1, with the carbohydrate on N172 positioned just outside heptad repeat A (Fig. 1). Cleavage of the N172 mutant was also reduced, and the level of overall expression detected following immunoprecipitation and SDS-PAGE was consistently less than that of the wild type (Fig. 5B and 7B). As expected, F1 of N172 runs faster on a reducing gel than F1 of wild-type F protein. F1 of N353 is not visible by SDS-PAGE (Fig. 5B and 7B), suggesting that cleavage of the N353 mutant is significantly impaired, which could result if the protein was not properly folded. However, all three N-glycosylation mutants were detected by biotinylation on the cell surfaces (Fig. 5B), suggesting that the change in structure affected availability of the cleavage site but was not so great as to cause retention of the protein in the endoplasmic reticulum.
Fusion promoted by each of the N-glycosylation mutants was decreased compared to wild type fusion; however, the extent of the reduction depended on which carbohydrate was removed (Fig. 7C and D). Syncytium and reporter gene assays of fusion were generally in agreement, although some variability was observed for N172 in the reporter gene assay. Consequently, the reporter gene experiment was repeated seven times, and 95% confidence intervals were calculated (Fig. 7D). N57 promoted fusion only slightly less efficiently than the wild type, while fusion promoted by N172 was greatly decreased. The extent of fusion promoted by these mutants may be a reflection of the level of proteolytic processing combined with the level of surface expression (Fig. 5B). As expected, fusion promoted by N353 was very poor. However, a few small syncytia could be found (Fig. 7C), suggesting that at least a small portion of the mutant F protein can be cleaved and promote fusion. However, the N353 mutant did not promote significant fusion above background levels in the reporter gene fusion assay (Fig. 7D).
The work presented here represents the first analysis of membrane fusion promoted by the F protein of HMPV, a recently identified paramyxovirus. We have shown that low-pH treatment of cells transiently transfected with only the HMPV F protein profoundly enhances cell-cell fusion. Indeed, a gradual increase in fusion was observed as the pH to which HMPV F-expressing cells were exposed decreased. Our results indicate that proteolytic processing of the F protein is a stringent requirement for fusion. However, the HMPV attachment protein, G, like other pneumovirus attachment proteins, is not needed for HMPV F-promoted fusion. In addition, we have examined the amino acid sequence requirements for trypsin-independent cleavage of the HMPV F protein when transiently expressed in cells in the absence of other viral components, and we have shown that the HMPV F protein is N-link glycosylated at three sites, and each of these carbohydrates is important for expression, cleavage, and fusion to various extents.
Fusion promoted by the F proteins of all previously examined paramyxoviruses, with only one possible exception (43), takes place efficiently at neutral pH. In vivo syncytium formation by a viral fusion protein is not consistent with a requirement for low pH to trigger fusion, as fusion takes place at the plasma membrane, where the pH is neutral. Strain-specific differences in the ability of HMPV to form syncytia have been reported (20), and it is not known whether the strain of HMPV from which the F and G genes used in this study originated can promote syncytium formation. Thus it is possible that only some strains of HMPV require low pH to trigger F protein-mediated membrane fusion and that these strains do not promote syncytium formation. While our data demonstrating that low pH stimulates F protein-promoted fusion do not prove that HMPV enters cells via an endosomal, low-pH pathway, further investigation of the mode of entry is clearly warranted. It is possible that a viral protein other than G provides for HMPV in vivo what low pH provides in vitro. Barretto et al. showed that transient transfection of RSV glycoproteins leads to a lack of reproducible syncytium formation in HeLa cells, while RSV infection promotes efficient syncytium formation (3). For RSV glycoproteins, neuraminidase treatment of glycoprotein-expressing cells greatly increased cell-cell fusion. However, neuraminidase treatment of HMPV F- and G-expressing cells did not alter the requirement for low pH to induce fusion (data not shown), though it should be noted that the optimal pH for neuraminidase activity (approximately pH 5) was not employed, as this would make the analysis of pH effects on fusion difficult. It is also possible that strains of HMPV which form syncytia have F proteins which can be activated in the secretory pathway under certain circumstances, as was suggested for infectious bronchitis virus (12), since the pH of some secretory vesicles (49) is at the level found to trigger HMPV F-promoted membrane fusion in this study.
Recombinant HMPV lacking the G protein is highly attenuated in primates (4), but in cell culture deletion of the G protein has no effect on virus titers (7). The RSV G protein enhances binding of virus to cells but has no role in fusion after attachment (44). Our results demonstrate that HMPV F protein-mediated syncytium formation does not require the G protein. Indeed, quantitative analysis of HMPV F-promoted fusion indicated that coexpressing G could reduce fusion levels (Fig. 3B). It remains possible that the HMPV G protein attaches to a specific cellular receptor but that this receptor is expressed only in certain cell types. This theory may be supported by the fact that syncytium formation in BHK cells was enhanced by the G protein (Fig. 4). However, BSR cells, which serve as the target cell type in the reporter gene assay where G expression was found to inhibit fusion (Fig. 3B), were originally derived from BHK cells.
As with other paramyxovirus F proteins, the HMPV F protein is N-link glycosylated and these modifications differentially affect the production of a mature and fusogenic F protein. Fusion promoted by N-glycosylation-site mutants decreases in the order WT > N57 > N172 > N353 (Fig. 7C and D). The levels of cleaved F protein on the cell surfaces also follow this order (Fig. 5B), suggesting that the extent of fusion is at least partially a reflection of the amount of mature F protein on the cell surface. This result is different from that observed for the RSV F protein, which contains two N-linked carbohydrate additions in F2 and one in F1. Mutation of each of these glycosylation sites had little effect on cleavage or surface expression of the RSV F protein (51). However, removal of one carbohydrate in RSV F2 actually resulted in an increase in syncytium formation, while removal of the carbohydrate in F1 resulted in a significant decrease in cell-cell fusion (51). Thus, individual N-linked carbohydrates modulate fusion of the RSV F protein but facilitate proper folding of the HMPV F protein such that proteolytic cleavage of the HMPV F protein is diminished to various extents upon the loss of individual carbohydrates.
The cleavage site of HMPV F differs significantly from the cleavage site of RSV F, which is cleaved by furin at two sites 27 amino acids apart (19). Wild-type HMPV F is not cleaved by an endogenous protease when expressed in Vero cells, despite the presence of a sequence at the cleavage site matching the minimal requirements for furin recognition (R-X-X-R) (5, 30, 41). However, our results clearly demonstrate that proteolytic processing is needed for membrane fusion (Fig. 3A and 6B). Cleavage-site mutations reported to promote or enhance endogenous cleavage of the HMPV F protein (41) did not give detectable cleavage in our in vitro system in the absence of trypsin. These differences may be a consequence of strain variation, or they could be due to the method of expression. A recent report examining the strain of HMPV F used in our study and containing the serine-to-proline mutation at P2 found a low but significant level of F protein cleavage when attenuated recombinant virus with the mutation was used to infect Vero cells (5). In contrast, no cleavage or replication of the wild-type virus was detected. Biacchesi et al. also measured a much greater level of cleavage with a mutant containing the same amino acid changes as our furin mutant. We detected inefficient endogenous cleavage of this F protein mutant in the same cell type (Fig. 5A), suggesting that the presence of other virus proteins may affect cleavage.
We discovered that HMPV F protein-promoted fusion requires proteolytic cleavage combined with exposure to low pH in order to induce appreciable cell-cell fusion. This raises the possibility that HMPV may be unique among paramyxoviruses, which are primarily thought to enter cells upon receptor engagement at the plasma membrane. Furthermore, our data suggest that the HMPV G protein may have a negative impact on F protein-promoted fusion, indicating that this "attachment" protein may also have a distinctive role in the viral life cycle. An endocytic/endosomal route of entry for HMPV requires further investigation, but our results clearly demonstrate that there are major differences between the F protein of this recently identified paramyxovirus and the paramyxovirus F proteins that have previously been characterized.
We thank Ursula Buchholz (NIAID, Bethesda, Maryland) for providing the HMPV F and HMPV G gene constructs, Robert Lamb (HHMI, Northwestern University) for the pCAGGS-SV5 F and HN expression vectors, Karl-Klaus Conzelmann (Max Pettenkofer Institut) for the BSR cells, and Dan Noonan (University of Kentucky) for the use of his luminometer. We are grateful to Roland Hilgarth for his assistance and members of the Dutch lab for critically reviewing the manuscript.
This study was supported by a research grant from the March of Dimes.
Accepted 29 August 2006.
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Journal of Virology Oct 2006, 80 (22) 10931-10941; DOI: 10.1128/JVI.01287-06
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Finally, Dr. Raymond Cook, an English Literature professor who had a profound impact on an unfocused college freshman about to flunk out of school, turned 99 years old last week. Dr. Cook lives in Valdosta and while his body is a bit frail, his mind is as sharp as ever. I don't want to over-dramatize it, but I doubt I would be where I am today and doing what I am doing had I not crossed his path those many years ago. It is a reminder to our public schoolteachers that while our politicians try to undermine you at every level, you are making a difference in some young person's life. Please don't give up.


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Check that the taxi ID number is visible. This should be on the left hand side in the front.
Make sure you get a cab not a private car. Taxis have identifying information such as the name of the company and the phone number.


The above are some of the common categories that people have separated themselves into throughout time, and will continue to separate themselves into. For some strange reason humans have a need to divide and oppress. These systems always need to keep one group below another. Keeping people fighting each other so they don't turn into the real enemy, or the real problems.
Humans for some reason seem to have a difficult time without it. They have some pipe dream that if they can just be one group or another group together things will be ok, but history has shown that is hardly ever the case.
When you had first nations people, they battled amongst each other. Tribes in Africa battled against each other. Europeans battled against each other, Asians and other groups the same thing.
Then some people think well if we could just have a separation of races again things would be ok, but they won't. People always find reasons to hate and oppression one another. When it is not race vs race, it's ethnicity vs ethnicity. When it's not this it's Rich vs Poor. When these options are not available they use religion. If religion is not available then you often have politics, one party vs another. In a society where none of these factors are an issue, you often then have a society where it's male vs females and a culture where women are often dominated by men. History has shown us this, yet people still think that if they can just separate it will all be ok, but it never is. People always have to find a reasons to hate and oppress each other.
They always seek reasons to put others down. If none of these above factors are available, they would use hair and eye color to discriminate, or skin tones. People always find reasons to not get along, and to try to make others feel inferior. It's a historical fact. You have witch trials, Inquisitions, political turmoil, race wars, revolutions. There have been few if any societies in history that got along without any of these factors being a significant part of their history, growth and development.
People see problems in society and they always try to find a reason for it. Oh it's the democrats, liberals, name your political party. It's those religious nuts, or it's one religion that is responsible for the problems in society. In other cases they look to race as the answer to the problems. In other cases male vs female and if we only had a more female oriented world. The truth is these natural oppositions are often used and exploited by the rulers in society to keep the people in their place where they belong. Not trusting, not getting along, and dependent on structures that are often oppressive to the people living in them.
The truth even though you won't believe it. We are spiritual beings. We come here to have various experiences. You will likely be male and female in your various incarnations. You will likely practice many religions, marry many different people if you marry at all. You will likely be many different races, have various political views, and ethnicity's. You will even have various eye, hair and skin hues. Your goal will be to try to attain growth. Many never do, it all depends on what you came to this plane to experience or what you came here to do. There is one song, and we are all just notes, which change from time to time, but we are all apart of the same one song. Most never tune into this, and in our societies most probably don't even believe it, and the more things go wrong, the more they try to find the answers by looking at our differences than our similarities.
On a purely physical level, the divisions in society are used and developed to keep people apart, to keep them not trusting each other and to make it harder for them to come together to over throw the ruling classes. By keeping us at each others throats arguing about the micro problems of society, we don't focus on the macro the real big things that might actually bring change.
Some will spend their whole life focused on one little spec of existence, one little problem and they will never see the bigger picture. I have been lucky to have a glimpse and I realise that the change that we all so ferverently desire will not be achieved at this level. The change that we desire is something deeper, and to attain it we must be in tune with higher frequencies. We need to go after those greater laws, rules, universal truths, but many in one life phase will never grow out of what they were taught and live in that one sphere and never break out of it, and therefore never attain full growth, truth, which can bring about the changes we all seek and desire. Many can never let go enough to see outside the boxes that we have been placed in.
A lot of people feel that there are things wrong with the world, and gather to discuss what is wrong. You can see the various groups across the Internet, and in each group you can see some very consistent and repeating patterns, and you begin to realise that it's not going to change on this level. This is a good level to become aware of the world and that there are some problems in the world, but many fall short of looking for the bigger picture. They find one answer, or stop at one leg of the journey, get comfortable and never keep searching for greater truth or enlightenment.
I have learnt a lot about the world and society over the last few years via research, and my targeting. A lot of what goes on in society is deliberately planned, or set up. Very little happens outside of a control mechanism. The various little groups online mirror their offline counterparts and you begin to see how staged things truly are. You you realise that much of what you see being played out in the forums are getting played out in real life, that is also another reason why things don't change. The house is stacked towards the system for a reason, and the more we play their games, the greater chance we have of losing, and never bringing about the changes so many of us desire and hope for in society.
United we stand, divided they fall.


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Category Archives: graphic novels
Cult TV From The 60s And 70s – The Terrible Shows We Used To Love
Posted on 17/10/2020 by Jorge Sette under art, culture, graphic novels, TV shows
Even if the reader did not watch these shows when they first aired, there were reruns throughout the seventies, eighties, and nineties, and some of them can still be watched on cable TV and streaming services. Besides, most of them are available as DVD box sets.
Younger people may find it hard to believe we loved those shows. How could we stand the primitive and amateurish visual effects? How could we tolerate the bias against women, gays, blacks and other minorities? How could we sit still through the slow pace, and the lack of jokes and punch lines present every other second in today's sitcoms?
Well, those were more innocent times, we were naïve viewers, we couldn't anticipate the complicated nuanced plots, complex social analysis and great acting of shows like THE SOPRANOS, MAD MEN, BREAKING BAD or HOUSE OF CARDS. Those early shows were all we had back then, and the whole family gathered together in front of the bulky black and white TV set to watch them. Few families had color TV in the early seventies in Brazil. Besides, now and then, one of us would have to stand up and reach for the TV aerial to adjust it or pound on the top of the TV set to get the image to straighten up. Did I forget to say there were no remote controls either? Bad news for the couch potatoes.
These were the most popular shows among my friends in those days:
Lost in Space. This was by far the kids' favorite show. When I was older, my mother explained the reason we were so into that show was that it featured a well-adjusted, loving family confronting the tough obstacles an ominous Universe put in their way. Could be. The aforementioned family – the Robinsons (any reference to The Swiss Family Robinson, the novel by Johann David Wiss published in 1812, is not coincidental) – sets off to investigate conditions to colonize a planet near Alpha Centauri, due to the overpopulation on Earth in the inconceivably distant future of ….1997!
Their trip would last 4 years, during which time they would remain frozen in suspended animation. However, many other nations were working on similar projects, competing with the US. Therefore, the wicked and ambitious Doctor Zachary Smith (Jonathan Harris), who worked for the US project as a psychologist, was hired as an agent by one of these competing nations. He carries the responsibility of sabotaging the mission. The problem is Doctor Smith gets trapped in the spaceship, the two-story disc-shaped Jupiter 2, seconds before it takes off, being forced to leave Earth with The Robinsons. The extra weight veers the spaceship off its original course and they get inevitably lost. He will be a burden to the family and their pilot, Major West, as they face innumerable perils in space or on the alien planets they sometimes land on. Rumor has it the producers' plan was to feature the family patriarch Professor Robinson (Guy Williams, of Zorro fame) and his co-pilot Major Don West (Mark Goddard) as the stars of the show. But as the episodes developed, the focus shifted almost 100% to the adventures of Doctor Smith (played hilariously by Jonathan Harris), the male kid, Will Robinson (Billy Mumy), and their loyal Robot, who stoically took all the abuse heaped upon him by Smith. The trio simply stole the show. Smith was supposedly gay (and rather camp), but no one talked about it at the time, and there was never any fear that he could corrupt his young partner, Will. The visual effects were pathetic and look ridiculous by today's standards. The settings and monster costumes are incredibly amateurish and silly too. But we loved the show.
I dream of Jeannie. Everyman's sexual fantasy come true, there was never, however, a more innocent relationship between a master and his sexy slave than the one sustained by Astronaut Major Nelson (Larry Hagman) and the genie he finds imprisoned in a bottle on a desert island on the Pacific, setting her free.
Actor Barbara Eden, who played Jeannie, was at the height of her beauty and sensuality in those days, and, although the network decency guidelines wouldn't allow us even a glimpse of her navel, she must have stirred the hormones of many a teenager and young man. Major Nelson, though, seemed immune to her attractions. We, on the other hand, were too young for those kinds of feelings and sensations. Girls loved the little doll house Jeannie lived in inside the bottle, while boys had the time of their lives watching the problems she caused Major Nelson by timing the execution of her magic tricks, accomplished by blinking her eyes and crossing her arms, to whenever Doctor Bellows (Hayden Rorke), the space program psychiatrist, was around.
The Time Tunnel
The Time Tunnel. This show did what every school should be doing: teaching history in a fun and engaging way. This, of course, was far from the objective of the producers, who only came up with a clever premise to raise their ratings, without any noble educational purpose in mind. The show featured two scientists trapped in a time machine built by the US government in the shape of a tunnel, hence the title.
The machine gets out of control and the scientists cannot return to the present. Every episode would feature a story in which they'd land either in the future or the past. The episodes depicting important past events (the Second World War, the eruption of the Krakatoa and the sinking of the Titanic, among others) outnumbered the ones set in imaginary futuristic scenarios, which turned the series into great history lessons. We learned a lot from watching it, and had fun at the same time.
Batman. The most psychedelic show of the era, the 60s version of the Dark Knight was an explosion of color (for those who could afford color TV), unforgettable idiosyncratic nemeses (such as Catwoman, the Joker, The Riddler and The Penguin) and exciting fight scenes during which innovative onomatopoeic speech bubbles popped up on the screen (Pow! Plop! Bang! Craack!). The costumes worn by most characters were duly ludicrous and it never crossed our naïve minds that the whole thing was supposed to be a parody of the comic books. We took the adventures very seriously: the anthological death scene of Catwoman, falling from a tall building after dangling for a couple of tense minutes by the grip of Batman's heroic hands before plunging into the void, depressed the most sensitive kids of the time. We all loved Catwoman, she was sexy and fun, who cared if she was evil?
Many other shows of the time were also popular, such as Land of Giants, Bewitched and The Monkees. Television evolved a lot in more recent years, and I daresay some of the new shows have way more quality than many of the movies we watch in theaters. I was lucky those silly shows coincided with my childhood and early teenage years: I was able to enjoy them fully without any sense of shame or guilt.
What was your favorite TV show of the sixties and seventies?
Six of Batman's Most Dangerous Enemies
Posted on 24/11/2019 by Jorge Sette under art, comic books, culture, graphic novels, language, movies
Batman, The Dark Knight, is one of the most iconic graphic novel characters of all time. Created by artist Bob Kane and writer Bill finger in 1939, Batman's secret identity is Bruce Wayne, an American billionaire whose parents were murdered when he was only a boy. As a consequence, Bruce Wayne swore revenge on all the criminals of the corrupt-ridden Gotham, the fictitious city he lives in.
This world-famous DC Comics's superhero has had his adventures turned into a popular TV show in the 1960s and numerous successful movies ever since. Bats, as he's oftentimes called by some of his friends and foes, is up against a huge gallery of rogues – most of them mentally-disturbed individuals who develop idiosyncratic theme-related personas with corresponding crime styles.
We have selected six of Batman's most dangerous enemies to discuss in this post. We cover their main personality traits, objectives and modus operandi, highlighting a couple of prominent quotes and naming famous actors who have portrayed them in movies or television.
Joker: one of the Cape Crusader's scariest villains. A sadistic clown, with a disturbing grin, Joker was responsible for the murder of Jason Todd, Batman's sidekick Robin, and for crippling Barbara Gordon, Batgirl, who became a paraplegic. In the movies, Joker has been played by great actors such as Heath Ledger, Jack Nicholson and Cesar Romero. Joaquin Phoenix, in the movie Joker, has given one of the most original and dazzling performances of the villain. One of Joker's quotes: "In my dream, the world had suffered a terrible disaster. A black haze shut out the sun, and the darkness was alive with the moans and screams of wounded people. Suddenly, a small light glowed. A candle flickered into life, symbol of hope for millions. A single tiny candle, shining in the ugly dark. I laughed and blew it out."
The Riddler: Edward Nigma wants, more than anything else, to prove his intellectual superiority over Batman. He challenges the Bat by throwing in riddles, puzzles and word games as clues to the crimes he's either planning or is already executing. The Riddler has been played on the big screen by Jim Carrey (Batman Forever, 1995). Two of his riddles:
Q: What is the beginning of eternity, the end of time and space, the beginning of every end and the end of every race?
A: The letter 'E'
Q: What belongs to you, but is used by others?
A: Your name
Two-Face: Harvey Dent was a close friend of Bruce Wayne's and a former district attorney, defending Gotham City against its criminals. His life changed radically after being assaulted by Gotham City's infamous mobster Sal Maroni, who cast acid into Dent's face, disfiguring half of it. The incident heavily affected Harvey Dent's mental health, causing him to develop an obsession with duality and the number two. Of course, this makes for a very interesting and sophisticated character, always torn between good and evil. Two-Face has the habit of flipping a one-dollar coin, with one of its sides suitably defaced, to make decisions about the conclusion of his crimes: eg. to kill a victim or not. Actor Aaron Eckhart offered a very convincing rendition of the rogue in the 2008 movie The Dark Knight. A quote by this fascinating villain: You have broken into our hideout. You have violated the sanctity of our lair. For this we should crush your bones into POWDER. However, you do pose a very interesting proposition: therefore, heads, we accept, and tails, we blow your damned head off!
Poison-Ivy: Pamela Isley, a student of advanced botanical biochemistry, is an eco-terrorist. Her aim is to protect plants at all costs. She won't hesitate to kill humans to protect nature, using all her knowledge of poisons and toxins to achieve her goals. Pamela has a love/hate relationship with Batman. Actress Uma Thurman portrayed the hot red-head in the 1997 movie Batman and Robin. A couple of quotes by the villain:
So many people to kill… so little time.
It took God seven days to create paradise. Let's see if I can do better.
Catwoman: Selina Kyle is a lover of felines and their protector. She is sometimes portrayed as a burglar and jewel thief. In most stories, however, she is more of an antihero than a typical villain, as her love/hate relationship with Batman tends to blur the lines. Actress Michele Pfeiffer infused the role of Catwoman with a powerful dose of sensuality in the movie Batman Returns (1992). Julie Newmar (below) played the most popular and beloved Catwoman in seasons 1 and 2 of the 1960s TV show. A quote: Meow!
The Penguin: Oswald Cobblepot uses his nightclub, the Iceberg Lounge, as a front for his criminal activities. His ultimate goal is financial gain, as it suits a businessman. Always smartly dressed in a tuxedo and top hat, the Penguin makes use of his collection of umbrellas as deadly weapons. The character has been unforgettably portrayed by actor Burgess Meredith (below) in the iconic TV series of the 1960s. One of the man-bird's quote: [to Catwoman] You're Beauty and the Beast in one luscious Christmas gift pack.
In the comments section below, please share with us your thoughts on Batman and his rogue gallery.
By Jorge Sette
Writing Powerful Comic Book Characters
Posted on 12/08/2018 by Jorge Sette under art, comic books, culture, graphic novels, language, Mythology, storytelling
In his extraordinarily insightful and funny book, Understanding Comics: The Invisible Art, author Scott McCloud states that comics is (yes, the verb is used in the singular!) a 3000-year-old sequential art form. More precisely, he defines comics as "juxtaposed pictorial and other images in deliberate sequence, intended to convey information and/or to produce an aesthetic response in the viewer." In modern times, however, the medium has mostly been used to combine pictures and words in the telling of stories.
Since we are in the realm of storytelling, we must necessarily allude to the seminal works of Joseph Campbell (The Hero with a Thousand Faces) and Christopher Vogler (The Writer's Journey) to advise writers on how to go about creating strong characters. Given the limited space of this blog post, we will not be covering the visual aspects of comics here.
Comics – sometimes called graphic novels, when not serialized but released as a standalone volume – is a medium, not a genre. As a writer, you must therefore stick to the set conventions (again, beyond the scope of this post) of the style of the work you are producing: be it romance, satire, horror, sci-fi, fantasy or superhero story. The guidelines for character creation that follow apply to all of those genres.
Writers who populate their stories with archetypes resonate a lot more deeply with their audiences. Archetypes – as defined by the famous psychologist Carl Jung – are characters or energies that represent mental functions common to all human beings. They are part of what is known as the collective unconscious and are projections of the different parts that together constitute a complete person, though they tend to appear as individual characters in a story. Let's illustrate our analysis of the main archetypes used in graphic novels with examples from Marvel's popularSpider-Man: Season One.
The hero is the story's protagonist. It is through his/her eyes that the audience experiences the story. It's therefore essential to create engaging characters that the reader can identify with. An effective way to do that is to make the character well-rounded. Popular heroes tend to balance noble qualities with major flaws. It's essential to avoid passivity in heroes. They are more appealing when they proactively conduct their journey.
In Spider Man, this is, of course, the part of Peter Parker.
The herald is the character, institution, or event that announces the upcoming adventure; he/she anticipates the need for the hero to leave his normal world (or the stable situation he finds himself in) and go on a mission. The herald is the bearer of disturbing news. Wrongs must be righted and only the hero can take on this responsibility.
The article on the Vulture's sightings in New York, featured in the Daily Bugle, sets off the action in Spider-Man: Season One.
In Joseph Campbell's words, a mentor is usually represented by the wise old man or woman. But, of course, any character, of any age, can perform this function in the story. The mentor helps the hero out, by example or advice. He plays the hero's role model.
Uncle Ben plays the mentor in Spider-Man.
Here we have the enemy. The hero's arch nemesis. The personification of the hero's worst fears. It's said that the shadow may also reflect what heroes don't like about themselves, their dark side.
In Spider-Man: Season One, this function is obviously performed by The Vulture.
Threshold Guardians
Threshold guardians are gatekeepers who are always testing the hero on their progress towards their goal. They guard the doors that will allow the hero to enter a more evolved phase. They might be confronted head-on or have their energies tapped into by the hero. Sometimes they happen to turn into allies.
Flash Thomson, one of Peter Parker's bullying schoolmates, is undeniably a gatekeeper in Spider-Man: Season One. The editor-in-chief of the Daily Bugle, J. Jonah Jameson, is another formidable threshold guardian.
The Trickster
These characters challenge the status quo; they provide comic relief (which is necessary to break the otherwise unbearable tension of a suspenseful story). Tricksters put things into perspective.
Peter Parker himself can be thought of as a trickster hero in Spider Man.
It's important not to forget that the archetypes listed above perform key dramatic functions in any story. Therefore, two or more of these masks can be worn by the same character at different points in the narrative.
This is all we have time for today. Good luck with the creation of powerful characters in your graphic novel. Let us know if the advice above is useful to you.

redpajama_set_name: RedPajamaCommonCrawl


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R&D Features
Natural colors uphold clean label trend
Switch to natural colors must be done in authentic way, experts note
By Stephanie Cernivec
GNT's Exberry colors, derived from fruits and vegetables, are non-GMO, halal, vegan, organic compliant and kosher. (Image courtesy of GNT USA Inc.)
For years, consumers have been interested in clean beverage labels with ingredients they recognize and understand. Thus, it's no surprise that natural colors are in demand. However, what's not always obvious is how much the color of a beverage plays into its marketing — both on and off the shelf.
As the saying goes, "You only get one chance to make a first impression." For beverages, this is crucial because color can significantly affect the initial perception of taste, which leads to a consumer picking up the product or not, explains Christiane Lippert, head of food marketing for Lycored, Orange, N.J.
• Beverage Colors
• Natural Colors
• Clean Label
Off the shelf, social media usage among millennials is putting even more pressure on beverage aesthetics, she notes.
"To this end, consumers increasingly base their purchase decisions on the visual appeal of a product," Lippert explains. "They want to buy into an experience rather than just a product, meaning the marketing of items is critical."
Visual excitement creates an opportunity to share a "like-worthy experience," which appeals to connected consumers, adds Maria Jose Alarcon, product experience marketing manager for colors with ADM, Chicago.
"[These consumers] are also more comfortable voicing what they want and need from their food and beverage products," she notes. "We've heard them loud and clear: colors from natural sources are key."
In fact, more than 80 percent of new product launches worldwide include natural color, says Meghan Fox, marketing specialist for St. Louis-based Sensient Food Colors, citing 2019 data from Chicago-based market research firm Mintel. She adds that the clean-label trend has led to a nearly 50 percent increase in new product launches with natural colors in North America, according to Mintel data.
ORBIS designs reusable beverage pallets typically used by can and bottle manufacturers.
Today's consumers are demanding easy-to-read ingredient statements and showing increased interest in plant-based products, creating a need for botanical colors, Fox says.
Retailers are even supporting the move from synthetic to natural colors, notes Jeff Greaves, president of Food Ingredient Solutions LLC, Teterboro, N.J.
"Pressure from retailers such as Whole Foods, Trader Joe's and Kroger is pushing more customers to move from artificial to natural colors," he says.
The trend toward natural colors is sweeping the beverage industry, experts say. However, if there's one category that's least affected, it's traditional carbonated soft drinks (CSDs), says Alex Sirosky, technical service manager for Sensient Food Colors.
Citing 2019 Mintel data, he says "all-natural" product claims account for only 3 percent of new CSDs in North America. Nevertheless, Sensient offers a fruit juice option for brown shades and carrot-based colors for strawberry or cream sodas, when interest in the category arises.
However, caramel color is generally the colorant of choice in the CSD category, notes Brian Sethness, executive vice president of sales and marketing for Sethness Roquette, Skokie, Ill.
"Because of the low pH of most carbonated beverages, Class IV caramel colors are commonly used because of their acid stability," Sethness explains. "In addition to providing excellent reddish to brown hues, caramel color can enhance the foaming characteristics, mouthfeel and flavor of soft drinks."
Although caramel color has been a long-time favorite in the CSD category, it's still being impacted by trends for non-GMO and organic ingredients — especially in the tea category, Sethness says.
The company now produces 11 Class IV Non-GMO Project Verified caramel colors, he says. Even though Class I caramel colors can only reach a certain level of darkness, they have become the fastest-growing segment of caramel colors because of their ability to meet consumer demand for clean labels, he adds.
Authentic use of color
Although the demand is there, it's often not enough to simply switch from synthetic to natural colors. Experts note that natural colors must be used in the right way.
For instance, beverages with bold, bright colors are typically perceived as artificial, says Roland Beck, head of business unit colors for Darmstadt, Germany-based Döehler.
"When a company switches its portfolio to natural colors because of brand repositioning or the launch of a natural brand extension, the most successful strategy has been to use cloudy, more muted colors that make the final product seem more natural and closer to juice," he explains. "Another successful strategy we have seen in recent years is the front labeling of the composition of juice drinks, thus avoiding having to label colors as additives."
For instance, an apple/cherry juice uses the cherry juice to impart color, but because the product is already labeled on the front of the package as a mixed apply/cherry juice drink, there is no need to declare a color in the ingredient list, Beck explains.
Beck also advises keeping the color of a beverage as close to the naturally occurring shades of the foods in the product. For example, instead of using a bright synthetic blue for a blueberry drinkable yogurt, use a purple shade of black carrot, he explains.
Natural color challenges
Of course, making the switch from synthetic to natural isn't always easy. Synthetic colors generally are known for being cost effective; predictable in shade and performance; and stable under various processing and storage conditions, Sensient Food Colors' Sirosky says. These accomplishments are no small feat.
In contrast, natural colors have inherent characteristics that can limit their use as colorants in beverages, ADM's Alarcon says. These include low color strength; thermal, light and pH instability; incompatible solubility; organoleptic impact; and tendency to oxidize, she notes.
"A wide range of factors — including pH, packaging, base color, processing conditions, temperature and shelf life — can also impact stability and performance of naturally derived colors in a particular application," Alarcon says.
To address these issues, ADM offers a patented clear emulsion technology, which provides stability to natural colors in a wide range of beverage applications, she says.
"The technology enables us to transform intrinsic solubility of natural raw materials into easy-to-use oil-in-water and water-in-oil color emulsions to allow naturally non-polar colorants to be used in water systems and vice versa," Alarcon explains. "Our technology allows the mixture of two or more immiscible liquids, provides heat-light stable colors without opacity, and prevents separation during shelf life in finished applications."
Food Ingredient Solutions' Greaves adds that pH and stability challenges can be addressed by managing a beverage's formulation.
"Natural colors' shade can change with pH, so pH needs to be controlled," he explains. "Stability can be affected by ascorbic acid and metal ions. Ascorbic acid helps stabilize carotenoid colors like beta carotene, but excessive amounts destabilize anthocyanin colors like grape. Divalent and trivalent metal ions cause otherwise stable anthocyanin colors to fade rapidly. This can be avoided in unfortified products by using [reverse osmosis] or [deionized] water."
Another challenge that fruit- or vegetable-based natural colors present is their flavor impact on the finished product, says Jody Renner-Nantz, applications manager of science and innovation for DDW, The Color House, based in Louisville, Ky. As a solution, Renner-Nantz notes: "DDW has developed Amaize Red, an anthocyanin derived from conventional or organic purple corn with low flavor impact even at higher use rates for berry-like beverages."
To tackle unwanted flavor impact, Sensient offers its Pure-S range, which uses gentle purification technologies to reduce or eliminate offnotes, Sirosky says. This creates bold colors while maintaining pure and simple ingredients, he adds.
Arguably the most vexing challenge for natural color suppliers is creating a natural blue that withstands formulation challenges. This is especially difficult because blue from spirulina extract and green from sodium copper chlorophyllin are not approved for ready-to-drink beverages in the United States, DDW's Renner-Nantz says.
Yet, color suppliers continue to innovate with solutions. ADM, for instance, offers a patented acid, light and heat stable blue color derived from huito fruit juice, sourced from the Amazon region of Peru, ADM's Alarcon says. This development makes it possible for the company to create a range of natural shades of blue, green, purple and brown, she says.
Likewise, Sensient Food Colors offers a proprietary natural blue vegetable juice that is heat, light and acid stable, enabling the company to deliver a range of blues, purples and greens across various applications, Sirosky says.
Improvements and Innovation
Despite the challenges facing natural colors, they've come a long way in the past few years.
"Natural colors are now achieving a level of brightness and stability that, some years ago, was only thought possible with synthetic colors," says Christian Benetka Uher, business development manager for Döehler. "Having learned to better understand the structure of natural colors and their interaction with other ingredients in food and beverage systems, we are able to develop better color formulations for the final application."
Plus, by controlling the process from the farm to the final product, suppliers can reduce the cost-in-use of natural colors to be competitive with some synthetic colors, Benetka Uher says.
In addition to reducing cost, vertical integration allows full traceability of ingredients, which is beneficial for companies looking to follow clean-label guidelines, Lycored's Lippert says.
GNT USA Inc. adds that its vertically integrated supply chain provides manufacturers with a consistent and steady supply of crops, says Jeannette O'Brien, vice president of the Tarrytown, N.Y.-based company. GNT's Exberry colors are derived from fruits and vegetables grown by GNT's select group of farmers and are non-GMO, halal, vegan, organic compliant and kosher, she says. Some colors also are certified organic.
Most recently, GNT developed a new range of Exberry liquid and powdered red colors without sugar ingredients.
"They offer higher color intensities and contain just two raw materials, helping food and beverage manufacturers achieve shorter, cleaner ingredient lists," O'Brien says. "Available globally, the new reds deliver colors that are 50 percent more intense, which means they can be used in lower dosages to achieve the same effect, resulting in reduced cost-in-use."
One of Lycored's most notable innovations is natural colors that can extend the shelf life of a beverage by as long as 12 months, Lippert says. This is especially important for sustainable packaging, she notes.
"Many beverage manufacturers want to reduce the level of packaging used for positive impact on both costs and sustainability targets," Lippert says. "To do this, stable color solutions that will survive light and chiller cabinet displays are crucial to product shelf life." BI
KEYWORDS: beverage colors clean label natural colors
Stephanie Cernivec freelances for Beverage Industry, writing for the magazine's print and online components. She earned a Bachelor of Arts in English with an emphasis in publishing and technical communications from Illinois State University.
Clean-label natural colors star of new product development
Natural colors requests grow in line with clean label
Clean label influencing colors market for beverages


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Experience scenic sunset views and watch birds and marine life up-close while touring the calm waters of the protected and shallow mangrove waterways. Paddling over seagrass beds with our naturalist guides, you will see and learn about birds, marine animals and other species which live below and above the water's surface. You will learn about the diversity of birds that inhabit the Virgin Islands, including summer, winter, and year-round residents and their migration patterns. This tour is for those who love to watch and ID birds as well as those with a curiosity for what makes the Mangrove Lagoon Wildlife Sanctuary & Marine Reserve such a unique ecosystem.
No previous kayaking experience is necessary, so this tour is perfect for beginners or seasoned veterans alike. After a brief orientation, you will load into your sit-on-top kayak, which accommodates two people and is easy to paddle. You will paddle through mangrove passageways toward Bird Island. There, you will see the egrets and pelicans that are nesting in the trees. You will paddle around the island and then continue around Bovoni Cay until you return to the marina. Look overhead to see the birds flying to their evening roost and take in all the splendors of a beautiful Caribbean sunset.
Free beverage and instruction included.
Kayaking with expert instruction and interpretation by our guides. Perfect for beginners!